Dissociation kinetics of actinomycin D from individual GpC sites in DNA

Abstract

We have examined the kinetics of dissociation of actinomycin from GpC sites in several DNA fragments containing synthetic DNA inserts, by a variation of the footprinting technique. Complexes of the ligand with radiolabelled DNA fragments were dissociated by adding a large excess of unlabelled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the GpC sites located in different sequence environments. Actinomycin dissociates more slowly from GpC sites flanked by (AT)n than An · Tn. Within regions of alternating AT, TGCA represents a better binding site than AGCT, and CGCA is a better binding site than GGCA. GpC sites flanked by (AC)n · (GT)n present good binding sites; in this context, dissociation from CGCG is faster than from TGCA.